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C Picat

Publications and source records attributed to C Picat.

At least 19 recordsLinked to original sources
PubMed1995

Functional analysis of DNase-I hypersensitive sites at the mouse porphobilinogen deaminase gene locus. Different requirements for position-independent expression from its two promoters.

Porphobilinogen deaminase (EC 4.3.1.8; PBG-D) is the third enzyme of the heme biosynthetic pathway. In both human and mouse, the gene encoding PBG-D possesses two promoters, lying in close proximity. We have previously reported the mapping of six nuclear DNase-I hypersensitive sites at the PBG-D locus which could contribute to the regulation of the gene. In the present study, and in order to define all the elements necessary for a high level of expression and an integration site independence, we studied the pattern and the level of expression of a cloned PBG-D gene following integration into a host genome. The longest construct that we tested (12.5 kilobases) contained sufficient regulatory elements to promote expression levels similar to that of the endogenous gene, both in transgenic mice and in transfected cells. The overall contribution of individual DNase-I hypersensitive sites to the expression of the gene was then studied using a series of mutants that were stably transfected into mouse erythroleukemia cells. Two regions seem to play a critical role in the erythroid-specific expression of the PBG-D gene: the proximal promoter and a region situated at -1000 relative to the initiation site. Study of individual clones of mouse erythroleukemia cells revealed that the erythroid-specific expression of the gene was submitted to position effects in the absence of the upstream region, although the housekeeping transcription is not sensitive to such effects. The tandem arrangement of the housekeeping and tissue-specific promoters of the PBG-D gene raises some questions about the functioning of these two overlapping transcriptional units in erythroid cells. Previous data have suggested that in erythroid cells most of the transcripts initiated at the upstream promoter stop downstream of the first ubiquitous exon, between the two promoters. Here, we show that the deletion of a constitutive DNase-I hypersensitive site that is located in the region of the elongation block results in opposite effects on the steady state levels of housekeeping and tissue-specific RNA. This finding is consistent with the hypothesis that this region promotes premature termination of the housekeeping transcripts therefore preventing promoter interference.

Animals↗
PubMed1992

A simplified method for determination of specific DNA or RNA copy number using quantitative PCR and an automatic DNA sequencer.

Quantification of specific RNA or DNA molecules that are present in minute amounts in biological samples has previously been performed using PCR in the presence of an internal standard. We have adapted this concept by introducing several modifications that facilitate the quantification of the products and obviate the need for radioisotopes. After amplification, individual products are separated on sequencing gels and directly quantified using a fluorescent automated DNA sequencer. We describe two applications of this approach: the quantitation of minute amounts of bcr-abl hybrid mRNA from malignant cells and the determination of gene copy number in cells stably transfected with a plasmid bearing a chloramphenicol acetyltransferase gene.

Automation↗
PubMed1991

Genetic heterogeneity of the porphobilinogen deaminase gene in Swedish families with acute intermittent porphyria.

Acute intermittent porphyria (AIP) is an autosomal dominant metabolic disorder affecting the enzyme porphobilinogen (PBG) deaminase in the heme biosynthetic pathway. The highest prevalence of the disorder has been observed in Scandinavia, especially in northern Sweden (Lappland) where it occurs with a prevalence of 1 in 1500. Biochemical assays of the activity and concentration of PBG deaminase in red blood cells, haplotyping with 4 intragenic restriction fragment length polymorphisms (RFLPs) (MspI, PstI, BstNI, ApaLI) using the polymerase chain reaction (PCR) and screening for known base substitutions by oligonucleotide probes was performed in 28 Swedish AIP families. There was no close relationship between haplotype, biochemical findings (PBG deaminase activity, enzyme-linked immunosorbent assay [ELISA], and excess urinary excretion of delta-aminolevulinic acid or PBG), and a specific mutation. Three different haplotypes were identified. The haplotype 2/1/1/2 (MspI/PstI/BstNI/ApaLI; +/-/-/+) was found to be the most frequent among gene carriers (P less than 0.001). The disease segregated with the haplotype 2/1/1/2 in the 10 families originating from northern Sweden. All 28 families were screened for three known point mutations. Only one was found to carry one of these mutations. Thus, the genetic background of AIP is heterogeneous in Sweden.

Alleles↗
PubMed1991

Spectrin Rouen (beta 220-218), a novel shortened beta-chain variant in a kindred with hereditary elliptocytosis. Characterization of the molecular defect as exon skipping due to a splice site mutation.

The molecular defect responsible for the shortened beta-spectrin chain variant, spectrin Rouen, was identified by analysis of cDNA and genomic DNA of affected individuals after amplification by the polymerase chain reaction. Peripheral blood reticulocyte RNA was transcribed into cDNA and amplified using primers corresponding to the 3' end of beta-spectrin cDNA. Agarose gel electrophoresis of cDNA amplification products from affected individuals revealed the expected band of 391 bp as well as a shortened band of 341 bp. Nucleotide sequencing of the shortened cDNA amplification product revealed that the sequences corresponding to the penultimate exon of the beta-spectrin gene (exon Y) were absent. This result was confirmed by hybridization of a Southern blot of amplification products with a labeled probe specific for exon Y. Nucleotide sequencing of the proband's amplified genomic DNA corresponding to this region of the beta-spectrin gene revealed a mutation in the 5' donor consensus splice site of the intron downstream of the Y exon, TGG/GTGAGT to TGG/GTTAGT, in one allele. We postulate that this mutation leads to the splicing out or skipping of exon Y, thus producing a shortened beta-spectrin chain. To our knowledge, this is the first documented example of exon skipping as the cause of a shortened beta-spectrin chain in a case of hereditary elliptocytosis. The exon skip results in the loss of the 17 amino acids of exon Y and creates a frameshift with the synthesis of 33 novel amino acids prior to premature chain termination 14 residues upstream of the normal carboxy terminus of the beta-spectrin chain, giving a mutant beta-spectrin chain that is 31 amino acids shorter than the normal chain.

Amino Acid Sequence↗
PubMed1991

Molecular heterogeneity of acute intermittent porphyria: identification of four additional mutations resulting in the CRIM-negative subtype of the disease.

Four mutations of the porphobilinogen (PBG) deaminase gene that result in cross-reacting immunological material (CRIM)-negative forms of acute intermittent porphyria (AIP) have been identified by in vitro amplification of cDNA from patients and by cloning of the amplified products in a bacterial expression vector. One mutation is a single base deletion which causes a frameshift and which is expected to result in the synthesis of a truncated protein. Two other mutations consist of single base substitutions and lead to amino acid changes. The fourth mutation is a single base substitution producing an aberrant splicing and resulting in an mRNA which would encode a protein missing three amino acids. DNAs from 16 unrelated CRIM-negative AIP patients were screened for the presence of these four mutations, by hybridization with oligonucleotides specific for each of the mutations, but none of the four mutations was identified in additional patients. The results indicate that mutations responsible for CRIM-negative AIP are highly heterogenous.

Amino Acid Sequence↗
PubMed1990

Hereditary pyropoikilocytosis and elliptocytosis in a white French family with the spectrin alpha I/74 variant related to a CGT to CAT codon change (Arg to His) at position 22 of the spectrin alpha I domain.

We describe a white French family in which 12 subjects presented with hereditary elliptocytosis (HE) or hereditary pyropoikilocytosis (HPP). Eight of these subjects were shown to be heterozygous for a spectrin (Sp) alpha I/74 variant, as demonstrated by analysis of partial tryptic digestion fragments of spectrin. This abnormal peptide pattern was associated with a decreased ability of Sp dimers to self-associate. In this kindred, in which four generations were available for study, the clinical expression varied from mild HE to HPP with an intermediate status of hemolytic HE. The severity of the disease appeared to be correlated both with the estimated amount of variant Sp (42% to 65%) and the excess of Sp dimers found in the membrane (30% to 51%, with a normal value of 3.7% +/- 1.6%). Reassociation studies using isolated Sp alpha and beta chains from an affected patient and an unaffected control subject showed that the Sp alpha I/74 Kd abnormal tryptic peptide resulted from a defect in the Sp alpha chain. Partial amino acid sequencing showed that the Sp alpha I/74 Kd peptide resulted from cleavage at lysine residue 42 of the Sp alpha I/80 Kd domain. Knowledge of the exon/intron organization of the human alpha Sp gene allowed us to amplify by the polymerase chain reaction the second exon of the alpha Sp gene in total cellular DNA of the HPP proposita. The amplified fragment was subcloned and sequenced. We found a G to A base substitution in the 22nd codon (CAT for CGT), which changes the normal arginine to a histidine. Hybridization of amplified DNAs with allele-specific oligonucleotides corresponding to the normal and mutant sequences confirmed the presence of the mutation in six other HE and HPP members of the family. The identification of this mutation at the DNA level confirmed the transmission of the same molecular defect in Sp through four generations but with different patterns of clinical expression.

Adult↗
PubMed1990

Identification of the mutations in the parents of a patient with a putative compound heterozygosity for acute intermittent porphyria.

The molecular abnormalities responsible for acute intermittent porphyria were investigated in both parents of a girl who was retrospectively diagnosed as having a homozygous form of the disease. The mutations in the parents are different from each other and both of them correspond to previously identified G to A changes in the coding part of the porphobilinogen deaminase mRNA. These point mutations lead to the presence of a catalytically-defective but immunologically-reactive enzyme. Our results support the conclusion that the propositus girl may represent the first case of compound heterozygosity for acute intermittent porphyria alleles.

Acute Disease↗
PubMed1990

Two different point G to A mutations in exon 10 of the porphobilinogen deaminase gene are responsible for acute intermittent porphyria.

Two mutations of the porphobilinogen (PBG) deaminase gene resulting in cross-reacting immunological material (CRIM) positive forms of acute intermittent porphyria (AIP) have been identified by in vitro amplification of cDNA and cloning of the amplified products in a bacterial expression vector. Both mutations resulted from G to A transitions in exon 10 of the gene and produced arginine to glutamine substitutions in the abnormal protein. Expression of mutant cDNA in Escherichia coli reveals that one but not the other of these amino acid changes results in a striking decrease of the optimal pH of the mutated enzyme. One or the other of these two mutations accounted for the defect causing AIP in six unrelated patients among the eight patients evaluated with the CRIM positive subtype of this disorder.

Acute Disease↗
PubMed1989

The mouse porphobilinogen deaminase gene. Structural organization, sequence, and transcriptional analysis.

The porphobilinogen deaminase gene encodes the third enzyme of the heme biosynthetic pathway. This gene is expressed in a tissue-specific manner and gives rise to two isoenzymatic forms encoded by mRNA species differing in their 5' extremity. Recent studies in human demonstrated that the tissue-specific expression of the porphobilinogen deaminase gene is determined in erythropoietic cells, by the utilization of a specific promoter situated 3' to the housekeeping promoter used in other cell types. This results, through differential splicing, in the mutually exclusive presence of either exon 1 or exon 2 in mature mRNAs. Here, we report the cloning and sequencing of the porphobilinogen deaminase gene from mouse. The overall organization of the mouse gene is similar to that of the human one. In the housekeeping promoter, only a short stretch of homology is found including two potential Sp1 binding sites; in contrast, more extensive similarity appears in the erythroid-specific promoter including two motifs also found in globin gene, a CACCC box, and a recently described Ery F1 consensus binding sequence. We derived a set of single-stranded probes corresponding to different parts of the mouse gene to carry out a detailed analysis of the transcriptional unit in various cell types, using a run-on transcription assay on isolated nuclei. In liver cells, the first (non-erythropoietic) exon is more actively transcribed than parts of the gene situated downstream, suggesting that the elongation of transcripts is blocked within the 5' part of the first intron. In erythropoietic cells, the downstream promoter becomes activated; surprisingly, the initiation of transcription is also enhanced from the upstream (housekeeping) promoter and most of the transcripts initiated at the housekeeping promoter stop downstream of the first exon, between the two promoters.

Amino Acid Sequence↗
PubMed1989

A point mutation G----A in exon 12 of the porphobilinogen deaminase gene results in exon skipping and is responsible for acute intermittent porphyria.

We have determined the mutation in a patient with acute intermittent porphyria. The mRNA coding for porphobilinogen deaminase was reverse transcribed then the cDNA was enzymatically amplified in vitro. Upon sequencing of a polymerase chain reaction product of abnormal size we found that this fragment lacked exon 12 of the gene. We analysed a genomic fragment containing exon 12 and determined that the patient was heterozygous for a point mutation G A at the last position of exon 12. We propose that this base change is responsible for an abnormal processing of the mutant allele such that exon 12 is missing in the mature mRNA. The resulting aberrant mRNA encodes a truncated protein which is inactive but stable and can be detected using antibodies directed against the normal enzyme.

Acute Disease↗
PubMed1989

An efficient laboratory made apparatus for DNA amplification.

DNA amplification by the polymerase chain reaction (PCR) is a method capable of producing a selective and very high enrichment of a specific DNA sequence. Hence it seems to be useful in various fields from basic research to clinical applications. In order to automatize PCR we assembled for a very low cost a mechanical system designed to carry a test tube holder successively in three thermal baths set at the required temperatures for the reaction. Two examples of the use of this machine are given: (i) amplification of DNA of a particular subtype of acute intermittent porphyria; (ii) the detection of the chimeric c-abl/bcr message found in chronic myelogenous leukemia cells.

Base Sequence↗
PubMed1989

Tissue-specific splicing mutation in acute intermittent porphyria.

An inherited deficiency of porphobilinogen deaminase [porphobilinogen ammonia-lyase (polymerizing), EC 4.3.1.8] in humans is responsible for the autosomal dominant disease acute intermittent porphyria. Different classes of mutations have been described at the protein level suggesting that this is a heterogeneous disease. It was previously demonstrated that porphobilinogen deaminase is encoded by two distinct mRNA species expressed in a tissue-specific manner. Analysis of the genomic sequences indicated that these two mRNAs are transcribed from two promoters and only differ in their first exon. The first mutation identified in the human porphobilinogen deaminase gene is a single-base substitution (G----A) in the canonical 5' splice donor site of intron 1. This mutation leads to a particular subtype of acute intermittent porphyria characterized by the restriction of the enzymatic defect to nonerythropoietic tissues. Hybridization analysis using oligonucleotide probes after in vitro amplification of genomic DNA offers another possibility of detecting asymptomatic carriers of the mutation in affected families.

Acute Disease↗
PubMed1989

Molecular analysis of acute intermittent porphyria in a Finnish family with normal erythrocyte porphobilinogen deaminase.

Porphobilinogen deaminase, the third enzyme of the haem biosynthetic pathway, is encoded by two distinct mRNA species expressed in a tissue-specific manner from a single gene. These two mRNAs are transcribed from two promoters and only differ in their first exon. An inherited deficiency or porphobilinogen deaminase in man is responsible for the autosomal dominant disease acute intermittent porphyria. Different classes of mutations have been described at the protein level suggesting that this is a heterogeneous disease. In the present report, we describe the molecular abnormality responsible for a variant form of acute intermittent porphyria where the enzyme defect is restricted to non-erythroid cells. Upon cloning and sequencing the mutant allele of a patient from a large Finnish kindred, a single-base substitution within the 5'-splice donor sequence of intron 1 was found at the last position of exon 1 (CG----CT). The identification of this mutation allowed us to detect asymptomatic gene carriers among family members using in vitro amplification of DNA and hybridization of the target sequence to allele-specific oligonucleotides.

Ammonia-Lyases↗
PubMed1989

[Research on genetic abnormality in the hemolytic form of hereditary elliptocytosis with homozygosity for the spectrin alpha I/74 variant].

We have undertaken to identify the spectrin gene mutation in a patient with a severe hemolytic form of Hereditary Elliptocytosis with homozygosity for the spectrin alpha I/74 variant. This variant corresponds to the presence of a 74,000 peptide which is produced during mild tryptic digestion of spectrin by cleavage at the Arginine-39 of the alpha I/80,000 domain of the spectrin alpha chain (595 amino acids). We hypothesized that the alpha I/74 mutation would be closed to the cleavage site Arg-39. A genomic library built with the patient's DNA was screened with a probe corresponding to a fragment of the alpha spectrin gene. Two clones were isolated, one being of paternal, the other of maternal origin. The subclones obtained contained the alpha spectrin gene exons 2 and 3 which encode for the first 88 amino-acids of the spectrin alpha I domain. The sequences obtained did not show any abnormality. The implications of these results are discussed.

Anemia, Hemolytic, Congenital↗
PubMed1988

Prevalence of the 281 (Gly----Glu) mutation in hepatoerythropoietic porphyria and porphyria cutanea tarda.

The prevalence of the 281 (Gly----Glu) mutation in hepatoerythropoietic porphyria (HEP) was investigated by the use of hybridization with a synthetic oligonucleotide probe. The mutation was found in HEP-affected members of two unrelated families from Spain, but was absent in two other patients from Italy and Portugal who also had HEP. Moreover, this mutation was not detected in 13 unrelated cases of familial (type II) porphyria cutanea tarda.

Carboxy-Lyases↗
PubMed1987

DNA polymorphism of human porphobilinogen deaminase gene in acute intermittent porphyria.

A common two-allele MspI restriction fragment length polymorphism of the human erythroid porphobilinogen (PBG)-deaminase gene was investigated in 33 unrelated patients with acute intermittent porphyria (AIP) and 20 controls. The polymorphism was tightly linked (lod score 3.14; no recombinants) to the locus for AIP as identified by measurement of erythrocyte PBG-deaminase activity. The frequency of the polymorphism in the AIP patients did not differ significantly from that in the controls. No common polymorphisms for eight other restriction endonucleases were found in either group. In 30 of the AIP patients no crossreacting immunological material (CRIM) was produced by the mutant PBG-deaminase allele. The MspI polymorphism enabled each PBG-deaminase allele to be distinguished in subjects heterozygous for the polymorphism; thus a major gene deletion was excluded as the cause of the CRIM-negative mutation in all of the 18 families that contained an affected CRIM-negative individual heterozygous for the polymorphism. In suitable families, the MspI polymorphism provides a more certain way of identifying carriers of the AIP gene than current enzymatic methods and major gene deletions are unlikely to be present in more than a small proportion of the commonest type of AIP, the CRIM-negative form.

Acute Disease↗